The present invention relates to spatial control of signal transduction by a biologically active, soluble molecule that transduces a signal to a cell having a receptor for the molecule present on a surface of the cell. In particular, the invention relates to, but is not limited to, control of signal transduction related to cell differentiation and tissue regeneration. Soluble signals are key regulators of stem cell phenotype in vitro.
Development of most tissue types involves a complex interplay of multiple signals leading to controlled precursor cell differentiation into mature, tissue-specific cell types. For example, marrow-derived mesenchymal stem cells (MSCs) can be differentiated in vitro into osteoblasts5-13, chondrocytes7,10,13-15, myoblasts13,16,17, adipocytes8,10,13,18, neurons and endothelial cells13,23 via exposure to a variety of growth factors that are known to be present in large quantities during development of the corresponding natural tissue.
One such growth factor is vascular endothelial growth factor (VEGF). VEGF is an endothelial cell mitogen24 that is operative in angiogenesis24-26. in response to 0.5 nM VEGF, MSC populations can differentiate into endothelial cells—the principal cellular component of blood vessels13,23,27—Also, bone morphogenetic protein-2 (BMP-2) plays a key role in bone development28 and drives differentiation of MSCs into functioning osteoblasts in vitro9,11. Further, in response to 2-200 nM insulin-like growth factor-1 (IGF-1), MSC populations can differentiate into adipocytes.18. These and other examples, demonstrate that a single growth factor, presented in an appropriate manner, can guide MSC differentiation down a specific pathway.
A next step in regenerative medicine involves extending these cell culture protocols to three-dimensional biomaterial constructs to control cell activity during tissue formation. Ultimately, control over growth factor activity in a three-dimensional construct would allow for orchestrated growth of hybrid tissues or organs containing multiple cell types from a single stem cell precursor. This objective is, as yet, unachieved.
Spatial control over the presence of soluble signals, such as protein growth factors, plays an important role in providing appropriate developmental environments. Limb morphogenesis is a classic example, in which spatially distinct presentation of fibroblast growth factors (FGFs), sonic hedgehog (SHH) and Wnt7a allow for simultaneous generation of the multitude of cell and tissue types present in a functioning limb1. Tooth morphogenesis is another more specific example, in which patterns of FGF-4, SHH and BMP-2 signaling drive simultaneous differentiation of primitive epithelium and mesenchyme into ameloblasts and odontoblasts, respectively. These examples, and others, demonstrate a characteristic that pervades tissue growth and development: supreme control over local growth factor activity during precursor cell differentiation4. Similarly, approaches for regenerating complex tissues are likely to rely heavily on signals, such as growth factors, as inductive agents for precursor cells. The relevance of controlled growth factor signaling in natural development has led to significant early work in inductive factor delivery in tissue engineering applications, and the successes and limitations of these approaches are described in detail below.
In view of the importance of such signaling during natural tissue development, several strategies have emerged for delivering growth factors to tissues33. Unfortunately, no method has yet approached the level of complexity displayed in natural development of complex tissues. Traditional growth factor presentation approaches focus on embedding growth factors in plastic microspheres34-38 or suspending growth factors in highly hydrated gels39-41. While these approaches are useful in various biomedical applications, their application to tissue regeneration is limited. For example, traditional plastic microspheres do not provide a structural matrix for tissue ingrowth and are difficult to process into structural matrices while retaining growth factor biological activity. Likewise, hydrated gels are also non-ideal carriers, as growth factors typically diffuse out of the gel matrix rapidly, resulting in limited signaling. Furthermore, these carriers do not fully recreate developmental processes because they do not provide spatial and temporal control at a microscopic level when presenting growth factors to tissues.
Newer materials allow longer-term growth factor release—up to several months—within structural matrices that support tissue ingrowth. Newer plastic microspheres are engineered both to bind to cell surface receptors and to release soluble nerve growth factor, thereby creating aggregates in which growth factor signaling can be “programmed” during tissue development43,44. In addition, a method for gas foaming of porous plastic scaffolds allows growth factors to be incorporated and released at variable release rates of up to several months with excellent biological activity45-47. This method is flexible in the types of growth factors that can be included and has recently been extended to deliver multiple active growth factors48.
In another approach, active growth factors are covalently immobilized in a hydrogel matrix to locally contain growth factor for signaling and to limit diffusion. This approach has been successfully applied to VEGF51 and nerve growth factor52-54 signaling, and could in principle be applied to other growth factors that remain active when covalently immobilized on a two-dimensional substrate, including transforming growth factor β2 (TGFβ2)55 and epidermal growth factor (EGF)56. Covalently immobilized growth factors have also been engineered to enable cell-triggered growth factor release, again leading to longer term growth factor activity. Taken together, these approaches have been very successful in actively influencing cell activity within structural matrices during tissue regeneration.
Growth, development and regeneration of nearly all tissue types are dependent on the ingrowth of a functional vascular supply. The vasculature not only allows for transport of nutrients and wastes to and from a neo-tissue, but has also been implicated in regulation of developmental processes such as endochondral ossification (bone development)88,89. The importance of a vascular supply in tissue regeneration has provoked widespread interest throughout the tissue-engineering field, and several investigators are focused on engineering functional vascular tissue within material constructs. Natural vascular ingrowth typically proceeds via either vasculogenesis, which involves de novo formation of an endothelium from primitive blood islands, or angiogenesis, which involves de novo formation of new blood vessels from a pre-existing vascular supply26.
The early stages of each of these processes involve rapid proliferation and organization of endothelial cells into tubes, followed by vessel maturation. Based on these natural processes, strategies designed to induce blood vessel ingrowth for tissue engineering applications have primarily focused on patterning of endothelial cells into networks90 or on delivering angiogenic growth factors and/or endothelial cells to promote new blood vessel growth46,48,91,92.
Current delivery approaches are not readily extended to a realm of spatially controlled growth factor activity because of an inability to process heterogeneous materials with distinct microscopic depots for releasing growth factors or because of an inability to further process polymer chains that are covalently attached to active growth factors. These limitations make it difficult to orchestrate multiple activities (e.g. stem cell differentiation) within a single material construct, which is likely to be crucial in complex tissue engineering. Accordingly, the limitations in current growth factor presentation systems demand new approaches that allow for patterned growth factor activity.